European patent application EP 0 974 840 the (840 publication), published Jan. 26, 2001, describes a device and system that may be used with the present invention. FIG. 1 presented herein as adapted from the 840 publication shows a parallel multi-channel test strip 2. In it, measurement areas 4, 6 and 8 are provided. Upon introducing a sample, usually whole blood, at introduction port 10 and depressing a bladder 12 and releasing it, a partial vacuum draws the blood though channel 14 up to shared stop junction 16. The test strip also includes a bypass channel 18 which draws sample toward bladder 12 to alleviate negative pressure at the stop junction order to prevent overcoming the surface tension that pins the fluid in the measurement areas at the stop junction.
For PT measurements, it is important to stop the flow of sample as it reaches that point to permit reproducible “rouleaux formation”—the stacking of red blood cells—which is an important step in monitoring blood clotting using the present invention. The principle of stop junction operation is described in U.S. Pat. No. 5,230,866.
A test strip body is described as preferably produced from three layers. The elements above are formed by cutouts in intermediate layer 20, sandwiched between a top layer 22 and bottom layer 24. Preferably, layer 22 is double-sided adhesive tape. Stop junction 16 is preferably formed by an additional cutout in layer 22 and/or 24, aligned with the cutout in layer 22 and sealed with sealing layer 26 and/or 28.
Each cutout for stop junction 16 is preferably at least as wide as channel 14. A filter may optionally be used to cover sample port 10. The filter separates red blood cells from a whole blood sample and/or may contain a reagent to interact with the blood to provide additional information. A suitable filter comprises an anisotropic membrane, preferably a polysulfone membrane of the type available from Spectral Diagnostics, Inc., (Toronto, Canada). An optional reflector may be on, or adjacent to, a surface or layer of test strip 2 and positioned over the measurement areas. If a reflector is present, the device becomes a transflectance device.
Typically, in producing the test strip, reagent is bubble-jet printed onto areas 4, 6 and 8. The chemicals at each site are disclosed in the 840 publication as: 1) thromboplastin in area 4; 2) thromboplastin bovine eluate, and recombinant Factor VIIa in area 6 and 3) thromboplastin and bovine eluate alone in area 8. The composition in area 6 is selected to normalize the clotting time-of a blood sample by counteracting the effect of an anticoagulant, such as warfarin. The composition in area 8 is selected to partially overcome the effect of an anticoagulent. The bovine eluate (plasma barium citrate bovine eluate) is available from Haemotologic Technologies, (Burlington, Vt.); recombinant Factor VIIa from American Diagnostica, (Greenwich, Conn.). Thromboplastin, from Ortho Clinical Diagnostics, (Raritan, N.J.).
After printing, a sample port is cut in untreated polyester film such as AR1235, available from Adhesives Research, (Glen Rock, Pa.) and then laminated, in register, to the top of the double-sided tape after removing the release layer. A die then cuts the stop junction through the three layers of the sandwich. Finally, strips of single-sided adhesive tape such as MSX4841, available from 3M, (St. Paul, Minn.) are applied to the outside of the polyester layers to seal the stop junction.
Use of the test strip can be understood with reference to a schematic of the elements of a meter shown in FIGS. 2A and 2B (also adapted from the 840 publication), which contemplates an automated meter. Alternatively, manual operation is also possible. In that case, bladder 12 is manually depressed before sample is applied to port 10, then released. The first step the user performs is to turn on the meter, thereby energizing strip detector 30, sample detector 32, measurement system 34, and optional heater 36. The second step is to insert the strip. Preferably, the strip is not transparent over at least a part of its area, so that an inserted strip will block the illumination by LED 38 of detector 40. (More preferably, the intermediate layer is formed of a non-transparent material, so that background light does not enter measurement system 34.) Detector 40 thereby senses that a strip has been inserted and triggers bladder actuator 42 to compress bladder 12. A meter display 44 then directs the user to apply a sample to sample port 10 as the third and last step the user must perform to initiate the measurement sequence. The empty sample port is reflective. When a sample is introduced into the sample port, it absorbs light from LED 46 and thereby reduces the light that is reflected to detector 48. That reduction in light, in turn, signals actuator 42 to release bladder 12. The resultant suction in channel 14 draws sample through the measurement areas to the stop junction. For each measurement area 4, 6 and 8, a LED 50 and detector 52 pair is provided to monitor the light transmitted through the sample as it is clotting.
Analysis of the transmitted light as a function of time (as described below) permits a calculation of the PT time, which is displayed on the meter display 44 and any messages regarding test strip fitness or reliability. Preferably, sample temperature is maintained at about 37° C. by heater 36. Each such function is controlled by a microprocessor chip 54 controlled by software stored in programmable, read-only memory 56.
As described above, the detector senses a sample in sample port 10, simply by detecting a reduction in (specular) reflection of a light signal that is emitted by 46 and detected by 48. However, that simple system cannot easily distinguish between a whole blood sample and some other liquid (e.g,. blood serum) placed in the sample port in error or, even, an object (e.g., a finger) that can approach sample port 10 and cause the system to erroneously conclude that a proper sample has been applied.
To avoid this type of error, another embodiment measures diffuse reflection from the sample port. This embodiment appears in FIG. 2B, which shows detector 48 positioned normal to the plane of strip 2. With the arrangement shown here, if a whole blood sample has been applied to sample port 10, the signal detected by 48 increases abruptly, because of scattering in the blood sample, then decreases, because of rouleaux formation. The detector system 32 is thus programmed to require that type of signal before causing actuator 42 to release bladder 12. The delay of several seconds in releasing the bladder does not substantially affect the readings described below.
FIG. 3 depicts a typical “clot signature” curve in which current from detector 50 is plotted as a function of time. Blood is first detected in a measurement area at time 1. In the time interval A, between points 1 and 2, the blood fills the measurement area. The reduction in current during that time interval is due to light scattered by red cells and is thus an approximate measure of the hematocrit. At point 2, sample has filled the measurement area and is at rest, its movement having been stopped by the stop junction. The red cells begin to stack up like coins (rouleaux formation). The rouleaux effect allows increasing light transmission through the sample (and less scattering) in the time interval between points 2 and 3. At point 3, clot formation ends rouleaux formation and transmission through the sample reaches a maximum. The PT time can be calculated from the interval B between points 1 and 3 or between 2 and 3. The result is typically reported in terms of its “INR” (i.e.,International-Normalized Ratio). Thereafter, the blood changes state from liquid to a semi-solid gel, with a corresponding reduction in light transmission. The reduction in current (C) between the maximum 3 and endpoint 4 correlates with fibrinogen in the sample.
Measurements made on a whole blood sample using the strip yield a curve of the type shown in FIG. 3 for each of the measurement areas. The data from the curves for the controls (measurement areas 6 and 8) are used to qualify the data from the curve for measurement area 4. The measurement of sample from area 4 is validated only when measurements on areas 6 and 8 yield results within a predetermined range. If either or both of these control measurements are outside the range, then a retest with another test strip is indicated. Ageing or oxidization of reagents can potentially yield failing Control 1 an/or Control 2 tests.